Evidence of mutualism between two periodontal pathogens: co‐operative haem acquisition by the H mu Y haemophore of P orphyromonas gingivalis and the cysteine protease interpain A ( I np A ) of P revotella intermedia

Summary Haem (iron protoporphyrin IX) is both an essential growth factor and a virulence regulator of the periodontal pathogens P orphyromonas gingivalis and P revotella intermedia , which acquire it through the proteolytic degradation of haemoglobin and other haem‐carrying plasma proteins. The haem‐binding lipoprotein H mu Y haemophore and the gingipain proteases of P . gingivalis form a unique synthrophic system responsible for capture of haem from haemoglobin and methaemalbumin. In this system, methaemoglobin is formed from oxyhaemoglobin by the activities of gingipain proteases and serves as a facile substrate from which H mu Y can capture haem. This study examined the possibility of cooperation between H mu Y and the cysteine protease interpain A ( I np A ) of P r. intermedia in the haem acquisition process. Using UV ‐visible spectroscopy and polyacrylamide gel electrophoresis, H mu Y was demonstrated to be resistant to proteolysis and so able to cooperate with I np A to extract haem from haemoglobin, which was proteolytically converted to methaemoglobin by the protease. Spectroscopic pH titrations showed that both the iron(II) and iron(III) protoporphyrin IX– H mu Y complexes were stable over the pH range 4–10, demonstrating that the haemophore could function over a range of pH that may be encountered in the dental plaque biofilm. This is the first demonstration of a bacterial haemophore working in conjunction with a protease from another bacterial species to acquire haem from haemoglobin and may represent mutualism between P . gingivalis and P r. intermedia co‐inhabiting the periodontal pocket.